There is a tremendous commercial demand for new antiviral products with novel mechanisms of action and which target a broad spectrum of viruses. Most previous and ongoing pharmaceutical development programs involve screening for inhibitors of essential virus enzymes with comparatively little investment in drugs that modulate the host immune response to infection. In this proposal, we focus drug development efforts on the host side of the virus-host interaction in order to discover and develop new antiviral products with novel mechanisms of action that are more effective, less toxic, and less sensitive to virus escape through mutation. Briefly, we propose to implement a high-throughput screen to identify small-molecule agonists of the cellular RIG-I pathway. RIG-I is a double-stranded RNA helicase that functions as a cytosolic pathogen recognition receptor that is essential for triggering immunity to a wide range of RNA viruses. Our group has developed a cell-based screening platform that is based on the use of Huh7 cells that are stably transfected with a luciferase reporter gene under the control of the ISG56 gene promoter (Huh7-ISG56-Luc). ISG56 is activated by IRF-3, a RIG-I effector molecule. This platform is amenable to high-throughput compound screening in which RIG-I signaling is identified through quantification of luciferase activity. In Specific Aim 1, we will reduce to practice the existing cell line and methods for high-throughput screening of RIG-I agonists. This will include scaling to microtiter plates, optimizing positive and negative controls, and defining all assay parameters. In Specific Aim 2, we will identify a set of lead compounds through library screening, counter screens, and validation assays. This will include screening a 20,000-compound maximally diverse library for agonists of the RIG-I pathway. Compound hits will be counter screened for target specificity and cytotoxicity and validated for dose-dependent activation of Huh7-ISG56-Luc expression and the ability to induce endogenous ISG56 gene expression in native Huh7 cells. In Specific Aim 3, we define the antiviral and mechanistic actions of lead compounds to identify a list of validated compounds suitable for optimization and pharmaceutical development. These assays will include examining the antiviral effects of compounds on a variety of viruses, including vesicular stomatitis virus (VSV), New Castle disease virus (NDV), hepatitis C virus, West Nile virus, respiratory syncitial virus, influenza virus, and human immunodeficiency virus-1. We will also begin to evaluate mechanistic aspects of compound function by examining the effects of compounds on IRF-3 activation kinetics, including IRF-3 phosphorylation, dimerization, and nuclear localization, and their action on RIG-I target gene and interferon-stimulated gene expression. This cell-based system and the targeting of RIG-I constitutes a unique drug discovery strategy. Since RIG-I is essential for triggering immunity to a wide range of RNA viruses, this approach offers the promise of defining broad-spectrum antiviral compounds. PUBLIC HEALTH RELEVANCE: We will use a unique drug discovery strategy to identify potential new antiviral drugs that work by activating a faster, more potent immune response to fight off virus infection. Our goal is to identify drugs that will work on a variety of viruses, including influenza virus, hepatitis C virus (HCV), West Nile virus, and human immunodeficiency virus-1 (HIV-1). Because these drugs will activate the natural immune response, they are likely to be safer, more effective, and less prone to failure due to the ability of viruses to develop drug resistance.